Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

HISTOCHEMICAL STUDIES OF THE SECRETORY

PROCESSES IN BOVINE SALIVARY

GLANDS

A thesis presented in partial fulfilment of

the requirements for the

Degree of Master of Science

at Massey University

Mervyn John Birtles

1981

Abstract of a thesis presented in partial fulfilment of

the requirements for the Degree of Master of Science

HISTOCHEMICAL STUDIES OF THE SECRETORY

PROCESSES IN BOVINE SALIVARY

GLANDS

by Mervyn John Birtles

Salivary glands from 12 bovine animals were dissected,

weighed and sampled for histological examination . The total

salivary gland weight was positively correlated with body weight

but there were not normally consistent differences between the

weights of left and right glands . However, in animals that had

chronic re - entrant cannulations of the left parotid and mandibular

ducts, the ipsilateral glands were always lighter. The histological

features of salivary glands and the histochemical reactivity of

their secretory and duct cells were examined. Parotid gland

secretory endpieces were elongated and their individual cells

contained PAS+ve granules . These cells were shown by

immunohistochemistry to be the site of protein secretion and thus

were classified as proteoserous cells . Chronic parotid duct

cannulation in association with duct obstruction caused dilation

of the secretory endpiece lumens and degenerative changes within

the endpiece cells. Intralobular duct cells contained PAS+ve

granules which may be the secretory component that is associated

ll

with secretory IgA. Variable numbers of intrastriated duct cells

occured in the parotid glands of different animals and in retrospect,

this was found to correlate positively with the animals known

susceptibility to bloat. The parotid excretory duct contained

many goblet cells which contribute a small amount of mucosubstance

to the proteoserous secretion .

Secretory endpieces of the mandibular gland were composed

of mucous cells which were PAS, AY and weakly AB +ve and demilune

cells which were PAS and AB+ve as well as acidophilic and

pyroninophilic . Clumps of plasma cells were observed in the

intralobular connective tissue . The effect of obstruction of

chronic duct cannulation on the mandibular gland was to dilate

endpiece and intralobular duct lumens, cause degenerative changes

in mucous and demilune cells and increase the numbers of small

lymphocytes, PMN neutrophils and mast cells in the connective

tissues of the gland. By contrast with the excretory duct of

the parotid, that of the mandibular contained no goblet cells but

simply a stratified columnar epithelium.

Mucous cells of the sublingual gland were PAS+ve, AY+ve and

weakly AB+ve and arranged into long tubular endpieces. The demilune

cells contained abundant PAS+ve, AB+ve, AY-ve granules . Many plasma

cells were present in the connective tissue between the secretory

endpieces and around the intralobular and interlobular ducts . In

animals with chronic cannulations of parotid and mandibular glands

the ispilateral sublingual gland weighed less than the contralateral

gland.

The posterior tongue, soft palate, pharynx and the lingual

iii

aspect of the epiglottis contained extensive areas of glandular

tissue . The secretory endpieces consisted of a high proportion of

mucous cells and a few scattered proteoserous demilune cells.

The glandular tissue of the epiglottis contained abundant plasma

cells in the intralobular connective tissue.

Based on their histochemical reactivity the demilune cells of

the intermediate buccal glands produced a purely serous secretion .

In addition, the intermediate and dorsal buccal glands contained

many AB, AY and PAS+ve mucous producing cells.

The labial glands were small , scattered lobules of secretory

tissue found at the labial commisures . The glandular lobules

were composed of tubular secretory endpieces capped with large

proteoserous demilune cells which were AY- ve , PAS+ve , strongly

a c idophilic and pyroninophilic . Large numbers of plasma cells

were found in the connective tiss ues within and around the secretory

tissue .

iv

ACKNOWLEDGEMENT My thanks are due to Dr D.H. Carr, who acting in the capacity

of supervisor, provided much help and encouragement during the

preparation of this thesis . I would also like to thank Professor

R. E. Munford for his helpful criticism of the text, for making

departmental facilities available for this study and generously

allowing the use of his typewriter.

I wish to thank Dr W.T. Jones, Applied Biochemistry Division,

DSIR, Palmerston North for supplying the salivary protein band 4

antibody and for permissi on to include the photograph (Figure 3 . 7)

of gel electrophoresis of bovine salivary protein. I am indebted

to Mr M.P. Gurnsey, Applied Biochemistry Division, DSIR, for

valuable assistance in obtaining the tissue samples and for

s upplying background information regarding the animals used for

this study.

My appreciation is extended to Ms Irene Hall for her assistance

during tissue collection and in the histology laboratory.

For the sketch showing the gross anatomy of the bovine salivary

glands (Figure 1 . 1), I gratefully acknowledge the artistry of Mr W. G.

Keereweer .

Finally, my thanks are due to Miss F.M. Williams, who typed

the draught copies of the manuscript and also my wife who typed the

final copy and who supported my efforts during the term of this

study.

V

TABLE OF CONTENTS Section

ABSTRACT

ACKNOWLEDGEMENT

LIST OF TABLES

LIST OF FIGURES

Page

ii

V

X

xi

1. INTRODUCTION 1

1.1 THE GROSS ANATOMY OF THE BOVINE SALIVARY GLANDS 5

1.1.1 Parotid Gland 5

1.1.2 Mandibular Gland 5

1.1.3 Sublingual Gland 6

1 .1.4 Ventral Buccal Gland 6

1.1.5 Intermediate and Dorsal Glands, Labial Glands 6

1.1.6 Palate Glands 7

1 . 1 .7 Lingual Glands 7

1.1 . 8 Pharyngeal Glands 7

1.2 THE GENERAL ORGANISATION OF SALIVARY GLANDS 8

1.3 THE HISTOLOGY OF SALIVARY GLANDS 9

1 .4 THE CYTOLOGY OF SALIVARY GLANDS 10

1.4.1 Serous Cells 10

1.4.2 Mucous Cells 11

1.4.3 Intercalated Ducts 12

1.4.4 Intralobular Ducts 13

1.4.5 Excretory Ducts 14

1.5 THE COMPOSITION OF SALIVA -THE SECRETION OF WATER AND ELECTROLYTES 15

1.6 THE COMPOSITION OF SALIVA -THE SECRETION OF PROTEIN

vi

20

Section

1.6.1 The Secretion of Non-irnrnunoglobulin Proteins

1.6.2 The formation of Salivary Protein

1.6.3 Secretion of Glycoprotein (mucosubstance)

1.6 .4 Immunoglobulins in Bovine Salivary Secretions

1.7 SUMMARY

2. MATERIALS AND METHODS

2.1 ANIMALS

2 . 2 DISSECTION

2 . 3 SAMPLING AND WEIGHING

2 .4 FIXAT ION PROCEDURES

2 . 5 PARAFFIN WAX PROCESSING AND SECTIONING

2 . 6 STAINING AND HISTOCHEMICAL METHODS

2.7 IMMUNOHISTOCHEMISTRY

2 . 8 MICROSCOPY AND PHOTOGRAPHIC EQUIPMENT

3 . RESULTS

3 . 1 SALIVARY GLAND WEIGHTS

3 . 2 HISTOLOGY AND HISTOCHEMISTRY

3.2 .1 Parotid Gland

3.2.2 Mandibular Gland

3.3.3 Sublingual Gland

3 . 3.4 Posterior Tongue and Soft Palate

3.3 . 5 Epiglottis

3.3 . 6 Dorsal , Intermediate Buccal Glands

3.3.7 Ventral Buccal Glands

3.3.8 Labial Glands

vii

Page

20

21

23

25

30

31

31

31

32

32

33

33

34

36

37

37

37

37

40

40

41

42

42

43

43

Section

3 . 4 HIGH BLOAT SUSCEPTIBILITY COMPARED TO LOW BLOAT SUSCEPTIBILITY

Page

43

3.5 CANNULATED GLANDS COMPARED WITH NON-CANNULATED 44

3 . 5 . 1 Animal Number Seven 44

3.5.2 Animal Number Eight 45

3 . 5.3 Animal Number Nine 45

3 . 5 . 4 Animal Number Ten 46

4. DISCUSSION 47

4.1 SALIVARY GLAND WEIGHTS 47

4. 2 HISTOLOGY AND HISTOCHEMISTRY 49

4.3 CANNULATED GLANDS COMPARED WITH NON- CANNULATED 58

5 . CONCLUSION 60

5.1 BOVINE SALIVARY GLAND CLASSIFICATION 61

6. APPENDICES 64

6.1 SUMMARY OF THE NATURALLY OCCURING POLYSACCHARIDES 64

6 . 2 APPENDIX 65

6 . 2 . 1 Histochemistry of Salivary Gland Carbohydrates 65

6 . 2.2 Histochemical Methods for the Demonstration of Group III Polysaccharides 65

6 . 2 . 3 Methods for Sialic Acid Identification 68

6 . 3 APPENDIX 71

6.3.1 Haematoxylin and Eosin 71

6.3.2 Alcian Blue 71

6.3 .3 Toluidine Blue 71

6.3.4 Alcian Yellow 72

6.3.5 Periodic Acid Schiff 72

viii

Section Page

6.3 . 6 Alcian Blue/Haematoxylin and Eosin 73

6.3.7 Alcian Blue /Haematoxylin/Periodic Acid Schiff 73

6 . 3.8 Alcian Blue/Alcian Yellow/Haematoxylin and Eosin 73

6 . 3 . 9 Alcian Blue/Alcian Yellow/Haematoxylin/ Periodic Acid Schiff 73

6 . 3.10 Alcian Yellow/ Haematoxylin/ Periodic Acid Schiff 74

6.3.11 Methyl Green Pyronin 74

7 . BIBLIOGRAPHY 75

ix

Table

1.1

2.1

2.2

3 . 1

3.2

LIST OF TABLES Following Page

Mean composition of mixed and parotid saliva secreted during high rates of secretion in cow receiving adequate intakes of sodium chloride

Summary of experimental animals

Paraffin wax processing schedule

Salivary gland weights

Apparent correlation between bloat susceptibility and the numbers of granular intra-striated duct cells found in bovine parotid and ventral buccal glands

X

16

31

33

37

39

Figure

1.1

3.1

3.2

3.3

3 . 4

3 . 5

3.6

3.7

3.8

3.9

3.10

3.11

3.12

3 . 13

3.14

3.15

3 . 16

3 .17

LIST OF FIGURES Following Page

Anatomy of the bovine salivary glands

Allometric diagram of total salivary gland weight (grams) and body weight (kilograms) expressed as logarithms

Allometric diagram of left and right parotid glands expressed as logarithms (grams)

Allometric diagram of left and right mandibular glands expressed as logarithms (grams)

Allometric diagram of left and right sublingual glands expressed as logarithms (grams)

Allometric diagram of left and right ventral buccal glands expressed as logarithms (grams)

Photomicrographs of parotid salivary gland

Electrophoresis of bovine salivary protein on on 7.5% polyacrylamide gel showing separation into numbered bands

Photomicrographs of parotid salivary gland

Photomicrographs of intralobular ducts from the parotid salivary gland

Photomicrographs of the excretory duct of the parotid gland

Photomicrographs of the mandibular salivary gland

Photomicrographs of the mandibular salivary gland

Photomicrographs of the mandibular salivary gland

Photomicrographs of the sub lingual salivary gland

Photomicrographs of the sublingual salivary gland

Photomicrographs of the sub lingual salivary gland

Photomicrographs of the glandular tissue within the posterior region of the tongue

xi

5

37

37

37

37

37

38

38

38

39

39

40

40

40

41

41

41

41

figure Following Page

3.18 Photomicrographs comparing the secretory endpieces of the tongue and soft palate 41

3.19 Photomicrographs of the glandular tissue found in the epiglottis 42

3.20 Photomicrographs of the secretory endpieces of the epiglottis 42

3.21 Photomicrographs of the dorsal buccal glands 42

3.22 Photomicrographs of the intermediate buccal glands 42

3.23 Photomicrographs of the dorsal buccal glands 42

3.24 Photomicrographs of the dorsal buccal glands 4 2

3.25 Photomicrographs of the labial glands 43

3.26 Photomicrographs of the mandibular salivary gland showing cellular changes due to an obstructed duct cannula 46

xii